May, 1938
DIELECTRIC PROPERTIES OF SERUM PROTEIN SOLUTIONS
increment a t high frequencies, 4~,/g, which depends chiefly upon the volume and hydration of the protein molecules; (3) the critical frequency, vc, which depends chiefly upon the size and shape of the molecules and upon the viscosity of the solvent. Methods are presented for the determination of these quantities by measurements of the dielectric constants of these solutions with a radio frequency bridge. The frequency range covered a t present is from 25,000 to 2,500,000 cycles per second, and solutions with specific conductivities
1123
up to about 1 X mhos/cm. can be studied. Measurements upon a series of solutions of crystallized carboxyhemoglobin have yielded consistent and reproducible results, and give AEo/g = 0.33, Ae,/g = - 0.11, and vc = 1.9 X lo6 cycles/ sec., interpreted as indicating a dipole moment for the carboxyhemoglobin molecule of about 500 Debye units, on the basis of an orienting dipole with a molecular weight of 66,700. The method is now being applied to a number of other proteins. CAMBRIDGE, MASS. BOSTON, MASS.
8, 1938 RECEIVED FEBRUARY
[CONTRIBUTION FROM THE DEPARTMENT OF PHYSICAL CHEMISTRY, HARVARD MEDICALSCHOOL, AND THE RESEARCH OF PHYSICAL CHEMISTRY, MASSACHUSETTS INSTITUTE OF TECHNOLOGY, No. 397 1 LABORATORY
Studies of the Dielectric Properties of Protein Solutions. 11. The Water-Soluble Proteins of Normal Horse B Y JOHND. FERRY AND J. L. ONCLEY
The dielectric constant of a solution of dipolar ions affords an estimate of the dipole moment of the solute, and is an important quantity in the interpretation of the interaction of such a solute with electrolytes and other dipolar ions.3 Studies of the dielectric constants of solutions of amino acids, peptides, and other dipolar ions of low molecular weight4 have contributed greatly to knowledge of the physical chemistry of these substances. A method recently described6 now makes it possible conveniently to measure the dielectric constants of electrolyte-free solutions of proteins over a wide range of frequencies. Data for these multipolar ions of colloidal dimensions may be interpreted by considerations similar to those which hold for the smaller dipolar ions. I n addition, from the dispersion of the dielectric constant may be calculated the relaxation time of a protein in solution, which is related to the shape and size of the molecule. Studies with the well-characterized protein, carboxyhemoglobin, already have been reported. The present paper extends this work to certain of the proteins of blood serum. ( 1 ) A preliminary report of this investigation was presented to the Division of Physical and Inorganic Chemistry at the 94th Meeting of the American Chemical Society, September 6-10, 1937. (2) This investigation has been supported in part by grants from the Committee of the Permanent Charity Fund, Inc., and from the Farnsworth Fund, Harvard Medical School. (3) Cohn, Chem. Reu., 19, 241 (1936). (4) Wyman, ibid., 19, 213 (1936). (6) Oncley, THIS JOURNAL, 60, 1116 (1938).
The protein components of serum usually have been separated by fractional precipitation from concentrated salt solutions. They have been characterized by their chemical compositions, molecular weights, isoelectric points, acid and base binding capacities, and cataphoretic mobilities, and by various physical properties of their solutions. Albumins.-The albumins of serum are crystallized readily from ammonium sulfate solutions of concentration more than half saturated. Their molecular weight, as determined by osmotic pressure measurements in a variety of solvents, is about 73,000;s,7and ultracentrifugal studies, which give almost the same molecular weight, have shown that, whether several times recrystallized or not, they are essentially monodisperse.8 Although the serum albumins have the same molecular weight, they can be separated by fractional crystallization into portions of widely different solubilities in concentrated salt solutions? The chemical compositions of such fractions also have been shown to differ considerably with respect to content of carbohydrate and certain amino acids.1° (6) Adair and Robinson, Biochem. J . , 24, 1864 (1930). (7) Burk, J . Biol. Chem., 98,353 (1932). (8) (a) Svedberg and SjBgren, THISJOURNAL, 110, 3318 (1928); (b) von Mutzenbecher, Biochcm. Z . , 266, 260 (1933); (c) McFarlane, Biochcm, J . , 29, 407, 660, 1209 (1935). (9) Mrensen, Compf.rend. frav. lab. Corlsbcrg, 18, No. I(1930). (10) Hewitt, Biochcm. J . , 80, 2229 (1936); 81, 860 (1937).
1124
JOHN
D. FERRY AND J. L. ONCLEY
Vol. 60
Globulins.-None of the proteins of serum remove salt, concentrated by ultrafiltration when neceswhich are insoluble in half saturated ammonium sary, and electrodialyzed until a specific conductivity of ohm-’ em.-’ was attained. Protein concensulfate solution has thus far been crystallized. about trations were determined by drying aliquots at 105’ and Those which are precipitated by concentrations weighing the dry protein. Weights obtained in this way of ammonium sulfate less than half saturated, are still subject to a correction for moisture, but the magand are insoluble in electrolyte-free water, are nitude of this correction has not been satisfactorily estermed globulins. At least three globulins have tablished, and it is therefore not introduced a t present. The PH of each solution was measured electrometrically recently been separated and characterized by with the hydrogen electrode. the methods of isoelectric precipitation‘l and Pseudoglobulin.-Two different procedures were emcataphoretic mobility.’* Two of these globulins ployed in the preparation of the pseudoglobulin. are only very slightly soluble in such dilute salt The globulins of preparation 11, which had been presolutions (of the order of N> as are a t pres- cipitated by half saturation with ammonium sulfate, were ent required for greatest accuracy in measuring dissolved in dilute salt and dialyzed against distilled water. Most of the protein was precipitated in this process, and the dielectric constants of protein solutions. was rejected. The protein now remaining dissolved was Investigation of the dielectric properties of these reprecipitated by half saturation with ammonium sulfate and redialyzed, the portions remaining dissolved at half globulins is therefore postponed. Pseudoglobulin.-If proteins of serum which saturation of ammonium sulfate (albumin’s) or precipihave been precipitated repeatedly by half satura- tated in the absence of salt (globulin) being rejected. The products electrodialyzed after three and four such repretion with ammonium sulfate are dissolved and cipitations appeared to have the same dielectric properties, subjected to dialysis and electrodialysis, pre- so the process was not continued further. In both cases, cipitation of globulin occurs, but a water-soluble the electrodialysis resulted in precipitation of somewhat fraction remains which may be and has some- more protein than could be removed by dialysis alone. times been called “pseudoglobulin.” Although The final solutions of “pseudoglobulin” were used for the measurements of dielectric constants. soluble in the absence of electrolytes, this “pseudoThe globulins of preparation 111, having been precipiglobulin” fraction resembles the globulins far tated by half saturation with ammonium sulfate, were fractionated further before dialysis. Only that fraction more closely than the albumins. Materials Normal horse sen.unl* was employed in three different preparations of the water-soluble proteins, albumin and pseudoglobulin. The first fractionation consisted in addition to the serum of an equal volume of saturated ammonium sulfate solution,which precipitated all of the globulins. Albumins.-After filtering off the globulin precipitate, as much of the albumins as possible WBS crystallized from the filtrate, following the procedure described by Sorensen for crystallizing egg albmin.14 The crystalline albumins of each preparation were then submitted to fractional crystallization in ammonium sulfate solutions of PH of about 5.1, the successive crops of crystals being treated separately. The three different preparations are designated by Roman numerals, and the successive crops of crystals from each preparation by the letters A, B, C, D. Each crop was recrystallized, the least soluble fraction A of preparation I1 as often as seven times. I n the case of IA and IIA, the first crop of crystals was suspended in an ammonium sulfate solution sufficiently dilute to dissolve the more soluble portions before recrystallization. The number of recrystallizations to which each crop was subjected is designated by a subscript. After recrystallization to the extent indicated, each albumin fraction was dissolved in water, dialyzed to (11) Green, J . B i d . Chem., 119, xxxix (1937); THIS JOURNAL, 60, 1108 (1938). (12) Tiselius. Biochcm. J . , 81, 1464 (1937). (13) We are much indebted to the Massachusetts Antitoxin Laboratory for the supply of this serum. (14) Siirensen, Compf. rend. trov. lob. Carlsbcrp, 11, 12 (1917).
soluble in 38% saturated ammonium sulfate was retained. This was precipitated a t half saturation with ammonium sulfate; the precipitate was dissolved in dilute salt and reprecipitated at half saturation, then dissolved and dialyzed against distilled water, atered, and a third time reprecipitated a t half saturation of ammonium sulfate. The portions soluble at half saturation of ammonium sulfate were always rejected.lK The final precipitate was dissolved, dialyzed against distilled water, concentrated by ultrafdtration to about 20% protein, and electrodialyzed. Although no protein precipitated upon electrodialysis, which yielded a solution of conductivity about and PH 5.2, fivefold dilution with conductivity water resulted in the separation of a greenish-blue, viscous gel.18 Greater dilution caused no further separation, and the solution was reconcentrated by ultrafiltration. The PH of this pseudoglobulin was 5.65 a t a concentration of 8.0 g. per liter. Further electrodialysis diminished the conductivity somewhat, but did not result in further precipitation or alteration of the dielectric constants of solutions of this fraction. This “pseudoglobulin,” which exhibits the same dielectric properties whether prepared by the above method or by that of preparation 11, may be constituted of more than one protein. (15) In the initial precipitation of the globulins by half saturation with ammonium sulfate, some albumin is carried down and can in fact be crystallized after re-solution. Most of the albumin may, however, be removed from the “pseudoglobulin” by several reprecipitations. (16) Addition of water to the gel changed it to a solid precipitate, presumably similar to the Pi of Green.” Dielectric studies on her PI, Pa, and Pa will be reported subsequently.
May, 1938
DIELECTRIC PROPERTIES OF SERUM PROTEIN SOLUTIONS
Experimental Results
1125
(IIAl), though the concentrations of the two solutions are nearly the same. The dispersion of the dielectric increment, however, lies in the same zone for both proteins. Both of these logarithmic dispersion curves are sigmoid and
The bridge method employed for measurements of capacitances and resistances, and the corrections involved in the calculation of the dielectric constant, have been described.s All measurements were carried out a t 25'. The original TABLE I capacitance and conductance data over the fre- DIELECTRIC MEASUREMENTS ON SERUMALBUMIN SOLUquency range from 0.025 to 2.5 megacycles for TIONS AT 25 FreConDitwo albumin solutions are given in Table I. Here quency ductance Polari- electric inmegapmhos, Capacitance zation K is the specific conductivity; A the empirical cycles, obsd., Obsd., Corr., corr., crement, G C cx -ACs Ae constant in the equation for polarization correction (in units of megohms"' ppf-''*); G1 the Serum Albumin IIAI, concentration 19.1 g./liter. X 10' = 11 mhos/cm.; A X 106 = 0.1426; initial conductance in parallel with the cell; GI 5000 pmhos; Co = 872 ppf. CQ the capacitance of the cell when filled with 1346 982 964 65 2.5 0.025 water; v the frequency in megacycles; G the 1348 962 945 45 2.5 .032 observed change in conductance (micromhos) 1350 948 931 32 2.4 .040 upon introduction of the cell containing the pro23 1352 940 922 2.5 .050 1355 933 916 16 2.5 .063 tein solution; and C the capacitance reading (in 1357 928 910 12 2.4 .080 micromicrofarads, ppf) of the standard conden1362 925 907 8 2.5 .loo ser. The capacitance of the cell, C,, is calculated 1367 922 905 6 2.4 .I25 from C by equations (9) of the previous paper,6 4 1373 919 901 2.3 .160 using the inductance constants given there. The 1382 917 900 3 2.2 ,200 1395 915 897 2 2.1 .250 polarization capacitance correction, ACs, is equal 1414 912 893 2 1.8 ,320 to AG2v-lJ. The dielectric constant increment, 1440 909 890 1 1.5 .400 DE, is given by the relation 1478 907 887 1 1.3 .500 1482 903 882 1 0.9 .630 A€ (Cx - AG2v-'.6- C0)/10.95 (1) O
Y
I(
=\
where the value of A is chosen so as to make A6 independent of frequency a t sufficiently low frequencies. Experimental justification for this procedure already has been given.6 The polarization correction term, AC6, is proportional to the square of the conductivity. I n our studies of each protein, there has been a considerable variation in conductivity and therefore in ACs, but not in the values of A E a t the same protein concentration. Whereas omission of the polarization term in (1) would appreciably increase values of AE calculated a t the lowest frequencies studied-at which the magnitude of AG2v-'J renders possible the most accurate estimates of A-this term diminishes with increasing frequency, and above one megacycle is entirely negligible. Albumins.-The data of the dielectric measurements on solutions of two fractions of serum albumin (each once recrystallized) are given in Table I, and the dielectric constant increments are plotted against the logarithm of the frequency in Fig. 1. At the lowest frequencies, A6 is represented as reaching a maximum, much higher for the albumin more soluble in ammonium sulfate (ICI)than for the less soluble one
.800 1.00 1.25 1.60 2.00 2.50 SerUm K
X
0.025 .032 .OM .050
.063 .080 .IO0
,125 .I60 .200 .250 .320 .400 .500 .630
.NO 1.00 1.25 1.60 2.00
2.50
1530 1594 1684 1803 1977 2249
900 897 896 896 902 911
878 874 869 865 864 862
0
0 0 0 0
0
0.6 0.1 -0.3 -0.7 -0.7 -0.9
Albumin IC1, concentration 21.7 g./liter. 12 mhos/cm.; A X 106 = 0.140; G1 = 5000 pmhos; Co = 880 ppf. 8.2 1058 1040 71 1417 1037 1019 49 8.3 1421 1424 1022 1004 35 8.2 8.2 1432 1013 995 26 1430 1006 988 18 8.3 8.2 1434 1000 982 13 995 977 9 8.1 1438 8.0 1446 992 973 7 7.9 989 971 5 1455 7.6 984 966 3 1465 7.1 979 960 3 1485 6.8 1509 975 955 2 6.2 968 948 I 1540 5.5 962 941 1 1585 4.8 1640 954 932 1 4.0 947 923 1 1714 3.2 941 915 0 1805 2.6 937 907 0 1928 1.7 933 898 0 2092 1.1 934 890 0 2316 0.7 939 885 0 2673
106 =
1126
JOHN
-
D. FERRY AND J. L. ONCLEY
nearly symmetrical about the point where v = 0.85 megacycles.
Vol. 60
different. The dispersion curves can be compared readily by adopting the dielectric increment a t v = 0.85 megacycle (which is the inflection point, vi, of the curves of Fig. 1) as a reference value, A q ; and plotting 1/2[1 (AEA t i > / ( A ~ - A e i ) ] against log v. Here Aeo is taken as the average value of AE a t frequencies of 0.1 megacycle and lower. It is seen (Fig. 2) that within the limits of error all of the measured points fall on the same curve, except for the repeatedly recrystallized preparation IIA,, which appears to be steeper than the others throughout; though the more purified preparations are all somewhat steeper a t high frequencies, where,
+
6
4
~~
-1.0 0 Loglo frequency in megacycles. Fig. 1.-Dielectric constant increments of serum albumin solutions plotted against the logarithm of the frequency: 1, albumin IC1, 21.7 g./liter; 2, albumin IIA1, 19.1 g./liter.
h \
IE
2
A t the highest frequencies, the dielectric constants of these solutions are lower than that of water, as has been reported by most previous investigators. The measurements do not yet extend to frequencies high enough to estimate values of AB,, which represent the situation Loglo frequency -1.0 in megacycles. 0 where rotation of the solute molecules adds nothing to the dielectric constant and their displacement of yater detracts from it. Fig. 2.-Dispersion of the dielectric conMeasurements upon four other albumin solustant increments of serum albumin solutions: 6,IIAI, 19.1 g./liter; C), IIA1, 50.6 tions are reported for all frequencies in Table 11, g./liter; 8, IC]; @, IA2; d ,IIIAs; P , IIA,. in which thec olumns for the uncorrected capaciFilled circles denote coincidences of two or tance C and the polarization correction ACg more points. are omitted to conserve space, since both are calculable from the equations and constants pre- however, our measurements are the least acviously cited. Data on a more concentrated curate. These differences are independent of solution of the albumin IIAl (cf. Table I) are the concentrations of the several solutions, which reported in columns 2-4. The other albumins for vary considerably. These dispersion curves, the which data are given have been recrystallized, shapes of which are subsequently considered, respectively, two, three, and seven times. The, represent quantitative descriptions of the dielecconcentrations of the various solutions are all tric properties of the proteins.
DIELECTRIC PROPERTIES OF SERUM PROTEIN SOLUTIONS
May, 1938
1127
TABLE I1 CONDUCTANCES, CAPACITANCES, AND DIELECTRICCONSTANT INCREMENTS OF SERUM ALBUMINSOLUTIONS Temperature 25"; GI5000 pmhos Fre-
quency v
0.025 .032 ,040 .050 ,063 ,080 ,100 ,125 .160 .200 .250 ,320 .400 .500 .630 .800 1.00 1.25 1.60 2.00 2.50 'K X ' K X K X K X
Albumin IA2 44.0 g. /literb
Albumin IIAi 50.6 g./liteP
-
CONDUCTANCES, CAPACITANCES, AND
Frequency Y
Albumin IIIAz 28.4 g./literc
cx At G Cr At G CS 962 2387 .4.9 1771 1126 7.5 1353 996 2392 4.9 1772 951 7.1 1355 1068 976 2398 942 1034 4.8 1782 7.0 1358 964 2401 936 7.0 1360 4.9 1783 956 1010 2407 5.0 1783 932 950 991 6.9 1362 929 944 2413 4.9 1785 981 7.1 1365 2421 4.8 1787 926 941 971 7.0 1369 2432 4.7 1791 937 962 923 6.8 1374 934 954 2446 4.6 1797 920 6.5 1382 6.2 1392 2465 917 4.4 1805 931 948 2488 927 940 5.7 1406 4.1 1816 914 2527 921 931 5.0 1427 3.6 1833 910 4.3 1456 2575 3.2 1854 917 922 905 2634 910 3.5 1492 2.7 1883 900 912 2671 2.1 1924 901 904 2.5 1539 896 896 891 1.6 1597 2754 1.4 1977 891 882 0.9 1667 2858 888 0.7 2042 885 2999 .1 1757 873 880 .1 2127 878 867 - . 4 3216 1877 2251 872 - .7 872 3496 2074 860 -1.1 867 -1.2 2425 868 3967 2283 862 858 -1.5 862 -1.6 2692 lo6 = 20; A X los = 0.120; co= 872ppf; Aeo = 7.0; Aei = 1.4. lo6 = 11; A X lo6 = 0.116; C0 = 8 8 0 p p f ; AEO= 4.9; Aei = 1.2. 10' = 14; A X loB = 0.054; C0 = 875 ppf; Aeo = 4.2; Aei = 1.2. lo6 = 18; A X lob = 0.0968; Co = 874 ppf; Aeo = 3.1; Aei = 1.1. G
G
Pseudoglobulin I1 15.0 g./liter"
cx 1056 1041 1026 1015 1005 993 983 972 959 947 936 924 913 904 894 886 880 875 869 866 862
AC
Ac
4.0 4.2 4.2 4.2 4.2 4.2 4.1 4.0 3.9 3.7 3.5 3.1 2.7 2.3 1.9 1.4 0.9 .3 - .3 - .7 -1.2
G
Albumin IlA7 31.4 g./literd C.
A6
2214 2217 2222 2224 2226 2229 2232 2236 2242 2251 2263 2282 2304 2334 2377 2437 2511 2610 2752 2955 3273
1030 993 967 950 939 929 923 919 915 912 909 905 902 898 89 1 887 883 876 869 862 857
3.3 3.3 3.1 3.0 3.1 3.1 3.1 3.1 3.1 3.0 2.9 2.6 2.4 2.0 1.5 1.2 0.8 .2 .5 -1.1 -1.6
TABLF I11 DIELECTRIC CONSTANT INCREMENTS OF SERUM PSEUDOGLOBULIN SOLUTIONS Temperature 25'; GI5000 umhos G
Pseudoglobulin 111
8.0 g./literb CX
Ac
G
8.5
/litera
Ex
A6
G
24.0
8~/literd
1023 11.6 1319 990 7.1 519 975 7.6 1036 1096 11.7 1322 1025 978 7.0 521 970 7.6 1041 1084 11.4 1324 1028 970 6.9 523 965 7.4 1047 1070 1328 1032 11.1 961 6.6 526 1053 959 7.1 1058 1037 1330 10.7 955 6.4 1060 529 955 6.8 1047 1044 1335 10.0 947 5.9 1072 533 949 6.4 1034 1053 .loo 1341 9.3 941 5.5 540 943 5.9 1085 1020 .125 1348 1065 934 5.0 8.4 547 1101 937 5.4 1006 1080 .160 1357 7.4 927 4.4 1121 555 929 4.7 991 .200 1097 1369 6.4 920 3.9 567 922 4.1 1144 975 1116 ,250 1381 5.5 913 3.3 579 915 3.5 1169 960 1141 ,320 1397 4.5 906 2.6 594 1201 908 2.9 944 1165 1413 .400 3.5 899 2.0 610 901 2.3 1233 930 1192 1433 .500 2.7 893 1.5 626 896 1.8 1268 917 1225 1457 ,630 1.8 889 1.2 627 890 1308 1.3 906 1261 .a00 1.1 1485 885 0.8 665 886 0.9 1352 897 1301 1.00 0.6 1518 883 .6 687 882 1402 .6 890 1.25 1356 .1 1564 880 .4 716 880 1467 .4 884 1431 1.60 1634 - .4 876 .o 756 877 .1 1561 879 2.00 1544 1745 - .7 875 .2 818 875 - .I 1684 874 1710 2.50 -1.1 1926 873 - . 3 909 873 - . 3 1868 870 ' Four times reprecipitated K x 108 = 8; A X lo6 = 0.21; C0 = 874 ppf; Aeo = 11.9; Aei 5.7 * Before final electrodialysis. K X lo8 = 11; A X IO6 = 0.083; C0 = 876 p p f ; Ac0 = 7.1; Aei = 3.4. K X 10' = 4; A X 108 = 0.240; c0 = 876 p p f ; AQ = 7.7; Aci = 3.6. K X lo6 = 9; A X lo6 = 0.173; Co = 876 ppf; Aeo = 16.3; Aci = 7.8. 0.025 .032 ,040 ,050 .063 ,080
-
-
A€
15.8 16.0 15.6 15.1 14.5 13.6 12.6 11.5 10.2 8.8 7.5 6.1 4.8 3.6 2.7 1.9 1.2 0.7 .3 - .2
-
.6
1128
JOHN
D.FERRY AND J. L. ONCLEY
Pseudoglobulin.-Measurements upon four pseudoglobulin solutions are reported in Table 111. Figure 3 shows the dispersion curves, reduced like those of Fig. 2 , with 0.24 megacycle as the frequency of the idection point, vi. In this case At0 is taken as 2% higher than Ae at 0.032 megacycle, where u/ui = 0.133, on the basis that the composite curve for the albumins shows A€/& cz 0.98 where v/vi = 0.133. The polari-
- 1.0
0
Log,, frequency in megacycles. Fig. 3.--Dispersion of the dielectric canstant increments of serum pseradoglobutin solutions: C), pseudoglobulin 11.4 times reprecipitated, 16.0 g./liter; @, 111, 8.0 g./ liter; O,III,8.5g./litw; 0,111,24.0g./liter. Filled circles denote coincidences of two or more points.
zation correction constant A is at the same time chosen to give the dispersion curve a t this point a slope appropriate to ”/vi = 0.133.l’ I n the plot of Fig. 3, all the data superimpose w i t h limits of error, regardless of the method of preparation or of the protein concentration. The resulting curve d&xs from that of serum albumin chiefly in that the dispersion occurs in a much lower frequency range. It is also wmmhat (17) The u n c a t i n t y involved in this choice is Of smsll maw?quence. Development of the apparatus for measurementl at lower frequencies will obviate such slight uncertainties in obtaining Aro when dispersion occurs in this region.
Vol. 60
less steep than the albumin curve. The curve of Fig. 3 may thus be considered a characteristic description of the protein fraction tentatively denoted “pseudoglobulin.”
Low Frequency Dielectric Increments The influence of proteins upon the static dielectric constants of solutions is given by the measurements a t low frequencies below the zone of anomalous dispersion. The values for the low frequency dielectric increment, A E ~ are , believed to be accurate to within 0.3 unit, and, as do the dispersion curves, serve to characterize proteins. Furthermore, as our studies on the serum albumins demonstrate, proteins with the same dispersion curves may have quite different dielectric constant increments (Fig. 1). Albumins.-Values of As0 estimated for the albumin solutions heretofore cited, as well as for certain others, data for the dispersion curves of which are not reported (since they are similar to those in Tables I and 11), are given a t a number of concentrations in Table IV. The PH and the conductivity of each solution are also reported. Although the dispersion curves of many of these solutions were closely similar, as noted, their didectric increments are very different. Values of A* for four albumin preparations are plotted against the concentration in Fig. 4 (curves 2-5). Over the range investigated, it appears that, as with hemoglobin,S and with amino acids and peptide^,^ the dielectric constant increases linearly with the concentration. The slope of TABLE IV LOW FRBQUBNCY DIELECTRIC CONSTANT INCREMENTS O F
ALBUMINSOLUTIONS Albumin
IIAi IAa
III&
IIAT
Ic1 XIID*
Concn., g./liter
19.1 50.6 13.0 26.2 44.0 15.2 24.3 28.4 51.4 31.4 66.2 11.8 21.7
6.8
9H
5.12 4.93 4.98 4.89 4.82 4.93
..
.. 4.81 4.80 4.68 5.40 5.20 5.23
12.3
*.
26.7
5.17 5.07
49.1
K
x
los 11 20 7
9 11 13 13 14 17 18 22 5 12
17 24 36 52
At0
A~Q/R
2.5 7.0 1.7 3.3 4.9 2.7 2.9 4.2 7.1 3.1 5.0 4.7 8.2 1.7 3.4 7.7 14.3
0.13 .14 .13 .13 .ll .18 .12 .15 .14
.10
.os .40 .38 .25
.28 .29 .29
May, 1938
DIELECTRIC PROPERTIES OF
each line in Fig. 4 represents the quantity Asa/g, which for each albumin studied is apparently independent of concentration, and is much greater for those fractions more soluble in ammonium sulfate than for those less soluble. A few measurements of solubility in ammonium sulfate solutions ($H about 5.1) were made on the fractions IC1 and IAt. The saltingout curves, following the equation of Cohn,18 log S = P-K:r/2, where S is the solubility of protein in g. per liter, and r/2 the ionic strength of ammonium sulfate per liter, may be described by the following constants: I C ~ ,p = 3.9, K: = 0.65; IAa, 3, = 5.6, K: = 1.11. The dielectric increment per g. per liter, AQ/g, is 0.39 for albumin ICl, which is about five times as soluble in ammonium sulfate solutions as albumin IA2 with AQ/g equal to 0.12. In another preparation, s k recrystallizations of a fraction of low solubility reduced the value of AeO/g from 0.14 (IIA1) to 0.10 (IIA,) , The data of Table I V include, besides the extreme cases illustrated in Fig. 4, fractions of intermediate dielectric properties. Pseudoglobulin.-Values of AQ estimated for the pseudoglobulin solutions heretofore cited, as well as for a number of others, are given in Table VI and are plotted as curve 1 in Fig. 4. In contrast t o serum albumin-which is crystalline and from some points of view apparently monodisperse, yet different preparations of which vary widely in solubility in salt solutions and in dielectric increment-the pseudoglobulin, which in some respects is not so well characterized a protein, appears to have much the same value of A 6 a t the same concentration, whether prepared as in preparation I1 or 111. Moreover, the influence of this fraction on the dielectric constant is much
than that of the albumins. Unlike the case of hemoglobin and the serum albumins, the cume for Aco against concentration (Fig. 4) indicates that the AcO/g of pseudoglobulin is nowhere independent of concentration. The value of A.Eg/g a t infinite dilution cannot be estimated with great accuracy, but it is approximately 1.1 per g. per liter, which is over ten times that of the least soluble serum albumin. Whether this fraction is monodisperse and what its molecular weight is remain to be investigated. It is possible that the constants reported for it may change somewhat with further purification.
0
TABLE V PSEUDOGLOBULIN SOLUTIONS Concn.,
g./liter
pH
11, 3 times repptd.
8.2
5.69 5.70 5.77
11, 4 times repptd.
17.6 32.0 4.4 8.2
15.0 28.0 111
a
x X
..
5.73 5.64 5.73 5.65 5.43 5.43
8.0" 8.6 24.0 Before final electrodialysis.
(18) Cohn, P k y s i d . Rcu., 6, 349 (192.5).
106
14 26 43 6 6 8
12 11
4 9
As0
Aco 1 fi
7.7 13.2 19.6 3.7 7.7 11.9 19.1 7.1 7.7 16.3
0.94 .75
.61 .84 .94 .79 .68 .89 .91 .68
1129
greater
Low FREQUENCY DIELECTRIC CONSTANT INCREMENTS OF Pseudoglobulin
SERUM PROTEIN SOLUTIONS
20 40 Concentration, g./liter. Fig. 4.-Low frequency dielectric constant increments. 1, Pseudoglobulin: '0, 11, 3 times repptd.; 9, 11, 4 timesrepptd.; 0,111. 2, Albumin IC,. 3,Albumin IIID2. 4,Albumin IAn. 5, Albumin IIAl. .
The dielectric increments per gram of the proteins thus far studied (0.10 for serum albumin IIA, to 1.1for serum pseudoglobulin) cover a similar range to that of the amino acids and peptides (0.17 for leucine to 1.3 for lysyl-glutamic acid). The dielectric increments of the proteins per mole are, of course, very much greater than those of the smaller dipolar ions. Taking the molecular weights for the proteins as 73,000 and 150,000, re~ p e c t i v e l y , ~the ~ ~ ~ dielectric ~' increments just
1130
JOHN
D. FERRY AND J. L. ONCLEY
quoted are per mole about 7000 for the albumin and 160,000 for the pseudoglobulin, as contrasted with 23 for leucine and 345 for lysyl-glutamic acid. Discussion Dipole Moments.-An estimate of the dipole moments of these molecules may be made from the empirical equation5 IA
= 2.9 d M (Aeo
-
Aem)/g
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experimental inflection point. If the dispersion for serum albumin and pseudoglobulin followed the Debye equation, the experimental points should fall on the calculated curves throughout, instead of only at vi where they are made to coincide. The deviations are actually small, though significant a t least for the pseudoglobulin. In any case, the reproducibility of the curves renders them very valuable for characterization of these proteins. Deviation from the theoretical curves might indicate that the simple Debye treatment is inapplicable to solutions of some proteins, even those composed of a single, symmetrical molecular species. For example, interaction between solute molecules with hindering of rotation might have the effect of spreading the critical frequency over a continuous range of values, which could produce slightly flattened dispersion curves following the experimental points of Figs. 2 and 3. On the other hand, the observed dispersion can be fitted by adding two or more components, each of which follows the Debye equation, so that
where ilf is the molecular weight. We shall assume Ae, = 2Aei - Act, (see below). For the serum albumins, the values are low or high according to the dielectric increments. For the albumin of lowest polarity IIA,, (AEO- Ae,)/g = 0.13 and p = 280 Debye units. For the most polar albumin, ICl, (Aeo - Ae,)/g = 0.42 and p = 510 Debye units. In the case of the serum pseudoglobulin, the ratio (Ae,, - Acm)/g is not independent of concentration. Its value a t idfinite dilution, as in the case of Aeo/g (1.1 per g./liter), can be estimated only tentatively. With the extrapolated value for (ACO- Ae,)/g of 1.2 per g./liter, p = 1200 Debye units. The dipole moments calculated for the two In this type of complex dispersion, the several albumins of most widely different solubilities in distinct critical frequencies, v ~ ( ~ )v,(~) , . . might salt solutions thus differ by a factor of about 1.8, be ascribed either to rotation of the same asymwhereas all the pseudoglobulin preparations have metrical molecule about different axes, or to the the same average moment, which is much higher presence of two or more symmetrical molecular than that of either albumin. While the values species. In the former case, analysis of the disestimated may be somewhat too large, they are persion curves should yield information about not probably much nearer the truth than figures cal- only the electrical but also the spatial symmetry of culated from the ordinary Debye formula,’* the molecule; the angles between its dipole mowhich is not valid for solutions in polar solvents. ment and its geometrical axes could be calculated. Dispersion.-The theory of the anomalous dis- If more than one molecular species be present, the persion of the dielectric constants of polar liquids shape of the curve should depend upon physical was developed by DebyeXg(pp. 77 ff .) ; it already chemical conditions (in the case of a reversible has been discussed in connection with protein so- equilibrium) or upon the state of purification (in lutions.a.m In the first paper of this series,6 the the case of a mixture of two unrelated proteins). dispersion for hemoglobin has been shown to folThe pseudoglobulin curve (Fig. 3) can be replow the equation of Debye resented on the basis of equation (3) by two dis( Aeo - Ae m ) v’/vC2 persion terms, with Ael = Aei, Ae2 = 2Aei - Aeo, Ae Aeo (2) 1 + v2/r2 VC(1) - 0.12 megacycle, and v,(~) = 0.48 megacywhere v, is the critical frequency. When v is cle; i. e., two Debye curves of approximately equal plotted logarithmically, the coijrdinates of the in- height, with one critical frequency equal to twice flection point are log vi = log v, and Aei = (Aeo the observed inflection frequency and the other A~m)/2. The form of this function is shown in equal to half the latter. Figs. 2 and 3 by dashed lines, drawn in each case The albumin data can be treated similarly, by obtaining vc and Ae, (= 2 A q - AEO)from the though in this case, since the most often recrystal(19) Debye, “Polar Molecules,” Chemical Catalog Co., New York, lized preparation appears to conform to the theo1929, p. 36. retical curve rather more closely than the others, (20) Williams, Trans. Faraday S O L ,SO, 723 (1934).
.
=I
+
May, 1938
DIELECTRIC PROPERTIES OF SERUM PROTEIN SOLUTIONS
there remains the possibility that further purification may yield a product whose dispersion shows no deviation from the simple Debye theory. Experiments to determine homogeneity from this point of view are in progress. Relaxation Times.-Serum albumin is supposed to be monodisperse from the point of view of molecular weights and electrical mobility.12 Recently reported values for the molecular weight range from 67,000 by the sedimentation velocity method21 to 74,600 by the osmotic pressure method.' On the basis of the estimatez2of 0.81 as the specific volume of isoelectric hydrated serum albumin crystals, the molecular volume of protein hydrate would not be greater than 80,000, as compared with 50,000 calculated from the lower molecular weight with the apparent specific volume of 0.74gEafor anhydrous protein in solution. Were this molecule symmetrical, the relaxation time in water a t 25' (given by r = 3qV/RT, where q is the viscosity and V the molecular volume) should be 0.055 X sec., or at a maximum 0.088 X 10-6. These values are only about a third of the figure 0.19 X sec. obtained from our measurements, taking as vc (= 1/2?rr) the inflection frequency of Fig. 2. Serum globulin is divisible into a number of fractions differing in isoelectric points, 11,12 solubilityJ1l electrical mobility,l2 and viscosity.23 The pseudoglobulin fraction prepared by the methods described above is being characterized further by all these methods. Despite the diversity in these chemical properties, the molecular weight of the main component of serum globulin has been given as 150,000 by ultracentrifugal studies21and 175,0OOs and 177,00024by measurements of osmotic pressure; while the ultracentrifuge also indicates a small component of molecular weight about 400,000. Relaxation times calculated from the smallest and largest of these values (in the absence of information concerning the molecular weight of the ''pseudoglobulin" we have used) would be 0.12 X and 0.32 X S ~ C . From our measurements, on the other hand, we have from the inflection frequency of Fig. 3 r = 0.66 X sec.; or, from the critical frequencies (21) Svedberg, Ckem. Reo., 10, 81 (1937); von Mutzenbecher and Svedberg, NuLur~,~ssenschoflcn, 11, 331 (1933). (22) Adair and Adair, Proc. Roy. Soc. (London), BllO, 422 (1936). (23) Unpublished experiments by A. A. Green and K. Fahey. (24) Burk, J . B i d . Chcm., 111, 373 (1937). (25) Adair and Adair22 give no value for the specific volume of hydrated pseudoglobulin. Their value for euglobulin is 0.81; although there is no justification for using this, i t would increase the calculated values of r by about 400j0.
1131
of the two Debye curves which are fitted to the data, 71 = 1.3 X l o p 6 and 7 2 = 0.33 X The discrepancies between the relaxation times calculated from the molecular weights and those observed are rather greater than in the case of the albumin (except that the r2 from the complex analysis corresponds fairly well to that calculated for the species of 400,000); whereas for hemoglobin the agreement appears to be sati~factory.~ While the dispersion of the dielectric constant, as reported, is an adequate quantitative description of each protein studied, the theory as thus far developed evidently does not permit calculation of molecular weights from observed critical frequencies. The extent to which asymmetry may account for not only the shape of the dispersion curves, but also their position at lower frequencies than anticipated, is being further investigated. In this connection it may be noted that the viscosities of solutions of hemoglobin, serum albumin, and serum pseudoglobulin deviate from the prediction of the Einstein equation increasingly in the order namedZ6-which is also the order of increasing deviation from the Debye equation, as regards both shape and location of dispersion curves. We are deeply indebted to Professor Edwin J. Cohn for his generous aid and indispensable advice throughout this investigation.
Summary 1. The dielectric constants of solutions of the pseudoglobulin and several crystalline fractions of albumin from normal horse serum have been measured over a frequency range of 25,000 to 2,500,000 cycles. 2. All these proteins increase the static dielectric constant of water. 3. The dielectric constant increments of those serum albumins more soluble in ammonium sulfate solutions are greater than those of the less soluble; the dielectric constant increment of the pseu~ ~ doglobulin is much greater still. 4. The dielectric constant increments per gram are of the same order of magnitude for these proteins as for the amino acids, and range from 0.1 for the least polar albumin to 1.1 for the pseudoglobulin. 5. The dielectric increments per mole range from 7000 for the least polar albumin to 160,000 for the pseudoglobulin. (26) Daniel and Cohn, THIS JOURRAL, 68, 415 (1926)
1132
M. A. JOSLYN
AND
6. The frequency dependence of the dielectric constant increment is the same for all the dbumins, deviating only slightly from a theoretical Debye curve with a critical frequency of 0.85 megacycle. The dispersion of the dielectric increment of all the pseudoglobulin solutions is given by a single curve which has a small but signifi-
C. MACKINNEY
Vol. 60
cant deviation from a Debye curve with a critical frequency of 0.24 megacycle. 7. The relaxation times calculated from these critical frequencies are several times larger than would be calculated from the molecular weights on the basis Of present theories* BOSTONMASS. cmBRIDGE,M ~
~
~RECEIVED . FEBRUARY 8, 1938
[CONTRIBUTION FROM THE FBUXT PRODUCTS DNISION, UNIVERSITY OR CALIFORNIA]
The Rate of Conversion of Chlorophyll to Pheophytin BY M. A. JOSLYN The conversion of chlorophyll to pheophytin, in dilute acid solutions, resulting in the replacement of magnesium with hydrogen, is a reaction which occurs a t a measurable rate in acetone solutions. Information on the rate of this r e a e tion is of interest because of possible contributions to an understanding of the complex linkage of magnesium in the phorbin nucleus. Willstatter and Stoll’ have shown that colloidal solutions of chlorophyll in water may be decomposed by carbon dioxide, and further that chlorophyll a is more rapidly converted to pheophytin a than the b component to the corresponding pheophytin b. Under comparable conditions for measuring the uptake of carbon dioxide, they observed, in forty-eight hours, 25 per cent. decomposition of chlorophyll b, as against 80% for chlorophyll a in twenty-four hours. These results were obtained by analysis of the magnesium contents of the partially decomposed chlorophylls. Dorries2 and Roben and Dorries3 have utilized the appearance in the pheophytin spectrum of a prominent band (which has a maximum absorption close to 5350 A. in acetone solution) as a qualitative means of detecting injury to leaves exposed to various gases. By observing spectrophotometrically the increased absorption at this wave length, we have estimated the proportion of chlorophyll converted to pheophytin in solutions of various acids in 90% aqueous acetone, at given time intervals. Our data substantiate the validity of this calx culation, where oxalic acid was used, because the initial rate was maintained for several hours, and (1) Willstatter and Stoll, “Assimilation der KohleusLure,” Verlag von Julius Springer, Berlin, 1918 (2) Dorries, Be? bot. Ges , 60b,47 (1932). (3) Roben and Dorries, i b r d , Sob, 52 (1932)
AND
G. MACEINNEY
we were able to demonstrate one chlorophyll and one pheophytin component by the Tswett column technique, in spite of the difficulties attendant upon the separation of components in solutions necessarily dilute for spectroscopic analysis. No other products were detected, so that the course of this reaction was reasonably established. Measurements were also made with sulfurous, sulfuric, and hydrochloric acids. The reaction rate was too slow with acetic acid for accurate measurement. The data may be interpreted on the basis of a first order reaction with respect to equivalent concentration of acid (i. e., normality). The data are less conclusive with regard to chlorophyll concentration. They favor a second order reaction for chlorophyll in oxalic acid solution, but with the other acids the initial rate is not maintained for a sufficient time for a satisfactory decision.
Experimental Stock solutions of chlorophyll were prepared by dissolving known amounts of chlorophyll (5X grade, American Chlorophyll Company) in acetone. Only preparations which responded satisfactorily to the tests listed by W&tatter and Stoll,‘ including the Molisch phase test, were used. Aliquots were pipetted into 2W-ml. volumetric flasks, diluted to m. 200 ml. with acetone, the appropriate amount of water and standardized acid added, and the solutions quickly brought to volume with acetone. The solvent was 90% aqueous acetone. The acid concentrations were varied from 0.05 to 0.0001 N , and the chlorophyll concentrations from 13.3 to 66.4 mg. per liter. The sulfurous acid solution was freshly prepared from liquid sulfur dioxide. Solutions were stored in the dark a t room temperature (20-25”), and periodic measurements were made. A Bausch and Lomb Universal Spectrophotometer was used to estimate visually the transmissions at the position (4) Willstatter and Stoll, “Untersuchungen fiber Chlorophyll ,“ Verlag von Julius Springer, Berlin, 1913,p. 144.
